Mycobacterium tuberculosis, the primary agent of tuberculosis, kills 2 million people annually, making it one of the world's leading causes of death. Moreover, it is a major cause of disease and death in AIDS patients, particularly in developing nations. Finally, multidrug resistant strains of M. tuberculosis (MDRTB) are potential agents of bioterrorism and have been designated Category C Priority Pathogens. Even in the absence of bioterrorism, the rapid global emergence of strains resistant to the major antibiotics used to treat tuberculosis poses a serious threat to public health. The highest priority in the fight against tuberculosis is the development of a vaccine that is more efficacious than the current vaccine - BCG. A vaccine more potent than BCG would have an impact on human health greater than virtually any other conceivable development in the fight against infectious diseases. During the previous grant period, we developed and characterized the first vaccine against tuberculosis more potent than BCG in a rigorous animal model. This vaccine, named rBCG30, is a live recombinant BCG vaccine expressing the M. tuberculosis 30 kDa major secretory protein. Compared with BCG, guinea pigs immunized with rBCG30 have significantly fewer tuberculous lesions in their lungs, livers, and spleens, significantly fewer M. tuberculosis organisms in their lungs and spleens, and significantly enhanced survival after aerosol challenge with highly virulent M. tuberculosis. rBCG30 was selected by the Aeras Global TB Vaccine Foundation and the Gates Foundation for further study in humans. In collaboration with Aeras, we successfully manufactured the vaccine by Good Manufacturing Practices (GMP) and completed studies required for an Investigational New Drug (IND) application from the Food and Drug Administration (FDA). rBCG30 is currently in human clinical trials. The guinea pig is highly susceptible to M. tuberculosis and develops a disease that closely resembles human tuberculosis clinically, immunologically and pathologically. Hence, it seems reasonable to postulate that the more potent a vaccine is in guinea pigs, the more potent it will be in humans as well. With this postulate in mind, and our having already developed a vaccine more potent in animals than BCG, the goal of this application is to develop a vaccine more potent than rBCG30 in the guinea pig model of pulmonary tuberculosis and ultimately, a vaccine that prevents or at least markedly reduces primary infection of the lung with M. tuberculosis. Lay Summary: Tuberculosis is a major worldwide public health problem. This proposal seeks to develop a more potent vaccine with which to combat this disease.